Many procedures employed in biomedical research and recombinant DNA technology rely on the use of labeled nucleotide or polynucleotide derivatives. In order for a modified nucleotide to be generally suitable as a labeled form of a naturally occurring nucleotide, several criteria must typically be satisfied. First, the modified compound must contain a substituent or probe that is unique (i.e., not normally found associated with nucleotides or polynucleotides). Second, the probe must react specifically with chemical or biological reagents to provide a sensitive detection system. Third, the analog must be a relatively efficient substrate for commonly studied nucleic acid enzymes, since numerous practical applications require that the analog be enzymatically metabolized (e.g., the analog must function as a substrate for nucleic acid polymerases). For this purpose, probe moieties should not be placed on ring positions that sterically or otherwise interfere with the normal Watson Crick hydrogen bonding potential of the bases. In such cases, the substituents can yield compounds that are inactive as polymerase substrates. Fourth, the detection system should be capable of interacting with probe substituents incorporated into both single stranded and double stranded polynucleotides in order to be compatible with nucleic acid hybridization methodologies. Fifth, the physical and biochemical properties of polynucleotides containing small numbers of probe substituents should not be significantly altered so that current procedures using hybridization probes need not be extensively modified. This criterion must be satisfied whether the probe is introduced by enzymatic or direct chemical means. Finally, the linkage that attaches the probe moiety should withstand all experimental conditions to which normal nucleotides and polynucleotides are routinely subjected (e.g., extended hybridization times at elevated temperatures, phenol and organic solvent extraction, or electrophoresis).
The specificity and tenacity of the biotin avider complex has been used in recent years to develop methods for visually localizing specific proteins, lipids, or carbohydrates on or within cells (E. A. Bayer and M. Wilchek, Methods of Biochemical Analysis, 26, 1, 1980). Chromosomal location of RNA has been determined by electron microscopy using a biotinized protein (e.g., cytochrome C) chemically crosslinked to RNA as a hybridization probe. The site of hybridization was visualized through the binding of avidin ferritin or avidin methacrylate spheres mediated by the avidin biotin interaction. (Manning et al., 1975, Chromosoma, 53: 107; Manning, 1977, Biochemistry, 61: 1364; Broker, 1978, Nucleic Acid Res., 5: 363; and Sodja 1978, et al., Nucleic Acid Res., 5: 383). This approach to the detection of polynucleotide sequences, although successful in the specialized cases examined, which were highly reiterated sequences, is not of general utility for analysis of polynucleotides present in single or low copy number.
Accordingly, there remains a continuing need for detectable labeled compounds that are safe, cost effective, stable, efficient, and provide for sensitive detection.